DESCRIPTION (provided by applicant: Microtubule-organizing centers (MTOCs or centrosomes play important roles in all eukaryotic cells. MTOCs are involved in mitotic spindle organization and in an increasing number of other fundamental cellular processes. Defects in MTOCs contribute to spindle defects, chromosome segregation defects, and genome instability. Additionally, MTOCs are the focal point for cytoplasmic microtubules, and are often near the nucleus of the cell. The structure and composition of these organelles is conserved among organisms. The animal and algal centrosome consists of pericentriolar material and a pair of cylinders called centrioles or basal bodies. The centrioles/basal bodies are critically important for ciliary assembly and function. In the last few years, studies from my laboratory have contributed to defining and understanding the complexity of basal bodies, to identifying critically important roles for two new tubulin family members in basal body/centriole function, and to recognizing that basal bodies serve as a complex signaling platform for sensory events in mammals. Basal bodies play an essential role in templating and docking proteins needed for flagellar/ciliary assembly. Basal body duplication in most cells is a precise event that gives produces exactly two new basal bodies each cell cycle. Using cryo-EM tomography, we will examine the role of a ring of amorphous material at the proximal end of the basal bodies, the growth of doublet and triplet microtubules, and the timing of the initiation of new basal body duplication. Second, we will investigate the role of basal bodies in recruiting and docking proteins needed for spindle function. Based on our observations, basal body integrity is needed to recruit the microtubule severing protein, katanin. We have found that a mutation in a NimA-like kinase, Cnk10, blocks katanin recruitment to the basal bodies. Cnk10 is the homolog of Nek1 that causes polycystic kidney disease in mice. To probe its role, we will find targets of this kinase in the basal body proteome using genetic and biochemical approaches. We will use immunoprecipitation with antibodies to katanin-tagged proteins to find basal body proteins that are needed for its docking. In a third project, we identified a protein (Stp2 that resides both in the basal bodies and the nucleolus. In the nucleolus, Stp2 is found in a doughnut-shaped structure. We will use genetic, biochemical and cell biological approaches to dissect the role of Stp2 in flagellar assembly and in regulating the density of cytoplasmic microtubules by studying two proteins identified by immunoprecipitation. We will also investigate the targets for its localization and its movement between the nucleolus and the basal bodies. In a fourth aim, we seek to identify genes that play a role in basal body/centriole maturation using a sensitized mutant (uni3 background. The maturation of centrioles/basal bodies during the cell cycle is likely to play important roles in both asymmetric cell division and signaling. PUBLIC HEALTH RELEVANCE: Microtubule-organizing centers (MTOCs are involved in mitotic spindle organization and in an increasing number of other fundamental cellular processes. Defects in MTOCs contribute to spindle defects, chromosome segregation defects, and genome instability. The centriole/basal bodies of the MTOC play roles in ciliary assembly and function and defects in cilia lead to loss of kidney function, retinal degeneration, obesity, hypogenitalia, infertility, cardiac malformations, neural tube closure, left-right reversals and embryonic lethality. We propose to use Chlamydomonas to study duplication and function of centrioles.